Application Note

Large Volume Direct Injection Method for the Analysis of Perfluorinated Alkyl Substances (PFAS) in Environmental Water Samples in Accordance with ASTM 7979-17

Kari L. Organtini, Gareth E. Cleland, Kenneth J. Rosnack

Waters Corporation

Abstract

In this application note we describe the use of the recently developed ASTM 7979-17 method (EPA Region 5,

Dr. Lawrence B. Zintek) to analyze PFASs of interest in environmental waters, not only as described by

U.S. legislation, but also those of interest elsewhere, including newer compounds (ADONA, 9Cl-PF3ONS, and

11Cl-PF3OUdS).

Benefits

Performing the ASTM 7979-17 method on the Xevo TQ-XS allows:

Limited sample preparation of small sample volumes to speed up analysis time and enhance sample

throughput.

■

Sensitive analysis of PFAS compounds in the low ng/L range to meet regulatory requirements.■

A robust and reliable solution for monitoring PFAS compounds in non-drinking water matrices.■

Introduction

Perfluoroalkyl substances (PFASs) are a class of anthropogenic compounds that are found in a range of

consumer goods and industrial processes due to their chemical properties. Common uses include firefighting

foams, insecticide formulations, water-resistant coating, floor polishes, and oilresistant coatings for paper

products approved for food contact. Due to their widespread use and subsequent leaching from materials,

PFASs are so ubiquitous that they are frequently detected throughout the environment and in 2009, they

were classified as persistent organic pollutants (POPs) within the Stockholm Convention.1 Due to their

persistent, ubiquitous nature, and possible toxicity, most regulatory agencies worldwide closely monitor the

use, occurrence, and impact of both traditional/common and newer, replacement short-chain PFASs.

For monitoring and research purposes, ng/L, or part-per-trillion (ppt), detection of PFASs is often required.

Within the U.S. drinking water is regulated under the Safe Drinking Water Act, while other environmental

waters are regulated under the Clean Water Act. In the third Unregulated Contaminant Monitoring rule

(UCMR3)2 for drinking water, the U.S. EPA has required monitoring of six different PFAS compounds with a

minimum reporting level in the range of 30 to 200 ng/L for each component. The U.S. EPA has also issued a

health advisory3 acute level at 70 ng/L based on the best available peer-reviewed studies of PFAS effects.

Within the EU, drinking water is regulated under the Drinking Water Directive, 98/83/EC, while other

environmental waters are regulated under the EC Water Framework Directive (WFD), 2013/39/EU.4 In the

WFD, PFOS is specifically identified as a “priority hazardous substance.”

In this application note we describe the use of the recently developed ASTM 7979-17 method (EPA Region 5,

Dr. Lawrence B. Zintek)5 to analyze PFASs of interest in environmental waters, not only as described by U.S.

legislation, but also those of interest elsewhere, including newer compounds (ADONA, 9Cl-PF3ONS, and

11Cl-PF3OUdS). Since many countries look to the U.S. EPA and other agencies for guidance, it was decided

to include as many compounds in a single analysis as was feasible at relevant detection levels.

Experimental

The ASTM 7979-17 currently covers the analysis of 21 PFAS compounds, with 10 additional compounds listed

for consideration in the appendix of the method. For this analysis, eight additional compounds were added to

the method to bring the total number of PFAS analytes to 39. Three of the compounds added to the method

are emerging PFAS compounds of interest including ADONA, 9Cl-PF3ONS (the main component of F-53B),

and 11Cl-PF3OUdS (minor component of F-53B). Table 1 contains the compound information for all of the

PFAS compounds included in this method. All standards were obtained from Wellington Laboratories

(Guelph, Ontario).

Table 1. PFAS compounds included in the analysis.

A Certified QC Standard (Cat #731) from ERA (Golden, CO), for use with ground and surface waters, was

used as an instrumental QC check throughout the analysis. The standard contained a mix of 12 PFAS

compounds. Certified values and QC Performance Acceptance Limits for each compound in the mix are

provided with the standard, making instrumental QC evaluation fast and straightforward.

Since required detection limits are in the low ng/L range and as a result of the widespread use of PFASs,

specific challenges must be addressed for sample collection, preparation, and analysis. There are many

common sources of PFAS contamination in the field and laboratory. In the field, caution should be taken to

avoid Teflon containing materials (such as waterproof clothing/jackets), plastic clipboards, waterproof

notebooks, and chemical ice packs. In the lab, items to avoid include sticky notes, certain glass disposable

pipettes, aluminum foil, vial caps with Teflon seals, and LDPE containers, to name a few. In fact, it is

recommended that all laboratory supplies be checked for PFAS contamination before use, as is practical.

Contamination is unavoidable from the chromatographic system. Therefore steps should be taken to

minimize any system contribution, and as such, the Waters PFC Analysis Kit (p/n:176001744) for the UPLC

system was utilized. The kit is comprised of PFAS-free components (such as PEEK tubing to replace the

conventional Teflon coated solvent lines) and a PFC Isolator Column that helps to delay any residual

background interferences from co-eluting with the analytical peak. Installation of the PFC Analysis Kit is

straightforward and quick.6

Sample pretreatment

Samples were provided by the U.S. EPA Region 5 through a Cooperative Research and Development

Agreement (EPA CRADA #884-16). Provided samples included reagent water, surface (river) water, ground

water, influent waste water, and effluent waste water. Each water sample was spiked with a low and high

level of a selection of PFAS compounds (three replicates of each concentration) prior to being received in the

lab. Two blanks of each sample were also received.

The entirety of each 5-mL water sample was used to avoid any compound loss. Each sample was spiked with

160 ng/L of isotopically labeled surrogates (see Table 1). The surrogates are added to the sample prior to any

preparation in order to determine method recoveries. 5 mL of methanol was then added to each water

sample and vortexed for 2 min. The entire 10 mL sample was filtered using a disposable polypropylene

syringe with a glass filter (25 mm diameter, 1.0 μm pore size) stacked on top of a polypropylene GHP filter

(25 mm diameter, 0.2 μm pore size). Following filtration, 10 μL of acetic acid was added to each sample. An

aliquot of each sample was transferred to a polypropylene autosampler vial and sealed with a Polyethylene

Cap (p/n: 186005230).

LC conditions

LC system: ACQUITY UPLC I-Class

fitted with PFC Kit

Column: ACQUITY UPLC CSH Phenyl Hexyl

1.7 μm , 2.1 × 100 mm

Column temp.: 35 °C

Sample temp.: 10 °C

Injection volume: 30 μL

Mobile phase A: 95:5 Water:methanol + 2 mM

ammonium acetate

Mobile phase B: Methanol + 2 mM ammonium acetate

Gradient:

Time

(min)

Flow rate

(mL/min)

%A %B

0 0.3 100 0

1 0.3 80 20

6 0.3 55 45

13 0.3 20 80

14 0.4 5 95

17 0.4 5 95

18 0.3 100 0

22 0.3 100 0

MS conditions

MS system: Xevo TQ-XS

Ionization mode ESI-

Capillary

voltage:

1.0 kV

Desolvation temp.: 500 °C

Desolvation gas flow: 1100 L/hr

Cone gas flow: 150 L/hr

Source temp.: 120 °C

Method events: Divert flow to waste from 15 to 21 min.

Method optimization using QuanOptimize

All MRM parameters for each compound were optimized using the QuanOptimize tool in MassLynx.

QuanOptimize will automatically determine the parent ion, fragment ions, cone voltage, and collision energy

required for each individual compound through injection. The MRMs generated from QuanOptimize for this

method are detailed in Appendix Table A. By providing the mass or chemical formula in the MassLynx

sample list, QuanOptimize will step through the cone voltages and collision energies designated in the

QuanOptimize method. The software then automatically processes the results and generates a report with

the MRM transition and corresponding cone voltage and collision energy (Figure 1). This tool also allows

rapid and simple optimization of MRM method parameters for new compounds that may need to be added to

the analysis method in the future.

Figure 1. Example of the MassLynx QuanOptimize tool showing the process of cone voltage and collision

energy optimization and the results from compound optimization.

Results and Discussion

Sample analysis was performed as described in ASTM 7979-17. One minor change was made to the mobile

phase composition. For this work, methanol was used in place of acetonitrile. Also, the concentration of

ammonium acetate added to both mobile phases was reduced to 2 mM from the suggested 20 mM in the

official method. Both changes were made due to solubility concerns of ammonium acetate in acetonitrile.

These changes had no negative effects on method performance, such as peak resolution or response but

made the LC method more robust. An overlaid chromatogram of all native compounds and isotope

surrogates is demonstrated in Figure 2.

Figure 2. Overlay of all PFAS compounds analyzed in the method.

Method Detection Limits

A Method Detection Limit (MDL) study was performed to assess the sensitivity of the sample analysis

method. Nine replicate samples were prepared by spiking reagent water with various concentrations of the

PFAS analytes (Table 2) and 80 ng/L of the surrogate standard solution. All samples were taken through the

sample pre-treatment procedure prior to analysis. MDL values were calculated using the equation:

MDL = SD x tn-1 , where SD = standard deviation of n replicates and tn-1 = 2.896 (student t value for n-1

samples)

The MDL values were all well below the required reporting limits stated in the ASTM 7979 method, indicating

that this method is more than suitable for this analysis. An MDL value could not be calculated for telomer

sulfonate isomer 6:2 FTS due to contamination of this compound in the solvents used for sample preparation.

The remaining PFAS compounds experienced no background interference or contamination. Calibration

curve requirements in ASTM 7979 require an R2 value of 0.98 or greater for linear regression fit. All

compounds were within this requirement, as highlighted in Table 2. Example calibration curves are also

shown in Figure 3 for PFOA and PFOS. Figure 3 also shows chromatograms for PFOA and PFOS injected at

2.5 ng/L. This demonstrates the sensitivity at half the required reporting limit for these two compounds.

Table 2. Method Detection Limit (MDL) results for all compounds in method.

*Reporting ranges listed are as set in the ASTM 7979-17 method.5

Figure 3. Example chromatograms and calibration curves for PFOA (top) and PFOS (bottom).

Chromatograms are representative of an injection of 2.5 ng/L, which is half the required lower reporting limit.

Control Samples

The ASTM 7979-17 method requires control samples to be run with the criteria they must pass as outlined in

Figure 4. All compounds passed the control criteria, with the exception of 6:2 FTS due to solvent

contamination of that compound.

Figure 4. Criteria set by ASTM 7979-17 method for control samples.

Sample Analysis

Triplicates of each spiked matrix at both concentrations were prepared and analyzed using the method

according to ASTM 7979-17. Only the compounds currently written into the ASTM method were spiked into

the water samples. All PFAS compounds that were spiked into the various water samples were detected at

both the high and low concentration spike. PFBA and PFPeA were spiked at 300 ng/L and 1000 ng/L in the

low and high spike samples, respectively. 4:2, 6:2, and 8:2 FTS were spiked at 1200 ng/L and 4000 ng/L in

the low and high spike, respectively. All other PFAS compounds were spiked at 60 ng/L in the low spike

samples and 200 ng/L in the high spike samples. Figure 5 shows an example of all the PFAS compounds

spiked at the low concentration level in the surface (river) water sample.

Recovery of the PFAS compounds was determined using isotope labelled surrogate standards that were

spiked into the samples prior to sample pre-treatment and analysis. For compounds that did not have a

surrogate available, a compound close in retention time and chemical structure was used as the surrogate.

Table 3 demonstrates the percent recovery of all PFAS compounds spiked into the five water samples. ASTM

7979 requires percent recoveries to be in the range of 70% to 130%. All compounds included in the method

were within this range with the exception of PFTreDA, PFTriDA, and FDEA. These compounds exhibited an

enhancement effect when analyzed from a prepared sample compared to the response exhibited in solvent

standards. The enhancement could be associated with co-elution of these compounds with matrix

components in the sample. Correction of sample concentration can be performed if necessary based on the

percent recovery exhibited by the surrogate standard or by quantification using matrix matched calibration

curves.

The method proved to be repeatable as well, demonstrated by the %RSD values highlighted in Appendix

Table B. All matrix samples were processed in triplicate, which is demonstrated by the n=3 RSD values.

These values represent the full method reproducibility. A single sample of reagent water and ground water

were also injected 20 times to produce instrument repeatability data (%RSD values in Appendix Table B).

Again, due to solvent contamination of 6:2 FTS, an accurate %RSD value for the method replicates could not

be calculated. For the most part, the %RSD values fell below 10%, with a majority of compounds exhibiting

RSDs below 5%.

Figure 5. All PFAS compounds detected in a low concentration spiked surface water sample. PFBA and

PFPeA at 300 ng/L; 4:2, 6:2, and 8:2 FTS at 1200 ng/L; all other compounds at 60 ng/L. *Compound shown

off scale.

Table 3. Percent recoveries of all compounds spiked into water samples.

Conclusion

The ASTM 7979-17 method allows for quick sample turnaround time due to minimal sample preparation.■

The results described here meet and exceed the ASTM 7979-17 method.■

The results described here meet and exceed the EPA health advisory acute levels of 70 ng/L PFOS.■

The large volume direct injection method used on the Xevo TQ-XS was extremely sensitive with method

detection limits in the low ng/L range for many compounds.

■

All targeted PFAS compounds were detected in the water samples analyzed at both low and high

concentrations with excellent recovery and reproducibility.

■

References

Secretariat of the Stockholm Convention. Stockholm Convention.https://chm.pops.int. Accessed 30

March 2018.

1.

U.S EPA. Third Unregulated Contaminant Monitoring Rule.https://www.epa.gov/dwucmr/third-

unregulated-contaminant-monitoring-rule. <https://www.epa.gov/dwucmr/third-unregulated-

contaminant-monitoring-rule.> Accessed 30 March 2018.

2.

U.S. EPA. PFOA & PFOS Drinking Water Health

Advisories.https://www.epa.gov/sites/production/files/2016-

06/documents/drinkingwaterhealthadvisories_pfoa_pfos_updated_5.31.16.pdf. Accessed 30 March 2018.

3.

Directive 2013/39/EU of the European Parliament and of the Council of 12 August 2013.https://eur-

lex.europa.eu/legal-content/EN/ALL/?uri=CELEX:32013L0039. <https://eur-lex.europa.eu/legal-

content/EN/ALL/?uri=CELEX:32013L0039.> Accessed 30 March 2018.

4.

ASTM D7979-17, Standard Test Method for Determination of Per- and Polyfluoroalkyl Substances in

Water, Sludge, Influent, Effluent and Wastewater by Liquid Chromatography Tandem Mass Spectrometry

(LC/MS/MS), ASTM International, West Conshohocken, PA, 2017, http://www.astm.org. <

http://www.astm.org./>

5.

L Mullin and J Burgess. Ultra Low-Level Detection of Perfluoroalkyl Substances (PFASs) Using the PFC

Analysis Kit. Waters Technology Brief no. 720005701EN. May 2016.

6.

Appendix Table A. MRM method details.

Appendix Table B. Reproducibility represented by %RSD of all compounds in each matrix. n=3 replicates represent the method reproducibility, n=20 replicates represent the instrument reproducibility.

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720006329, June 2018

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